This invention relates to polarized light and, more particularly, to enhancing polarized light microscopy.
A polarized light microscope system (“pol-scope”) has the ability to measure submicroscopic subjects such as molecular arrangements dynamically and non-destructively, e.g., in living cells and other specimens. For at least this reason, the pol-scope has been widely used in biological research, as described in U.S. Pat. No. 5,521,705 to Oldenbourg, et al., entitled “Polarized light microscopy”, (“the '705 patent”) which is incorporated herein by reference, and in the publications discussed below, which are incorporated herein by reference.
The use of video cameras to record images of birefringent specimens with the polarized light microscope was described by Allen and collaborators (Allen, R. D., J. L. Travis, N. S. Allen and Ho Yilmaz. 1981. Video-enhanced contrast polarization (AVEC-POL) microscopy: A new method applied to the detection of birefringence in the motile reticulopodial network Allogromia laticollaris, Cell Motil. 1:275-289) and by Inoué (1981. Video image processing greatly enhances contrast, quality and speed in polarization-based microscopy. J. Cell Biol. 89:346-356). To measure specimen birefringences from recorded images one can use a predetermined calibration curve to relate measured intensities to specimen birefringences in different parts of the image (Schaap, C. J. and A. Forer. 1984. Video digitizer analysis of birefringence along the length of single chromosomal spindle fibres. Cell Sci. 65:21-40). This method is relatively fast, but may be subject to errors from variations in light intensities that stem from other sources than birefringence, e.g., light scattering or shading. In a study on edge birefringence, Oldenbourg recorded images of a given specimen at several different compensator settings and used a stack of images to compute the specimen birefringences independent of the overall intensity and the background light in different parts of the viewing field. Oldenbourg, R. 1991. Analysis of edge birefringence. Biophys. J. Vol. 60 page 629.
With the traditional pol-scope, thus, single images display only those anisotropic structures that have a limited range of orientations with respect to the polarization axes of the microscope. Furthermore, rapid measurements are restricted to a single image point or single area that exhibits uniform birefringence or other form of optical anisotropy, while measurements comparing several image points take an inordinately long time.
The '705 patent describes a pol-scope that permits data collection and determination of anisotropic structures (e.g., specimen birefringence) irrespective of orientation and over a wide range of magnitude. The '705 patent pot-scope has the sensitivity to observe biological cells and tissue structures that are otherwise either difficult to see or require staining or labeling with exogenous dyes for adequate contrast. The '705 patent pol-scope allows measurement of the birefringence magnitude and the orientation of the birefringence axes in the specimen, thus providing information on the submicroscopic alignment of molecular bonds and fine structural form.
A principal axis of alignment, or birefringence axis, is determined in the plane perpendicular to a microscope axis. Methods are currently available for observing the birefringence component parallel to the microscope axis. One such method, called conoscopic imaging, is typically applied to single crystals. Another method uses a special universal rotation stage known as Fedoroff universal stage (A. N. Winchell, Elements of Optical Mineralogy, 1931, New York: John Wiley & Son, at least at pp. 60, 209) that includes two glass hemispheres encapsulating the specimen. Neither method is compatible with biological specimens which typically require immersion objectives for high resolution imaging (which is incompatible with the universal rotation stage) and which include many birefringent structures that are too faint to observe conoscopically.
In addition, with regard to industrial applications such as rock and soil analysis for oil exploration, a typical analysis of rock samples with a polarizing microscope is a labor-intensive process yielding the orientation of one micro-crystal at a time. A thin rock section is mounted on a special universal rotation stage and the sample is viewed under different tilt angles (Hartshorne, N. H. and A. Stuart. 1960. Crystals and the Polarising Microscope: A Handbook for Chemists and Others. London, Arnold.).
Further, the purity of the polarization is dependent on the characteristics of light extinction in the pol-scope. In general, the higher the extinction, the better the purity and uniformity of the polarization. Polarization distortions may result from reduced extinction caused by a high numerical aperture objective and condenser lenses of the microscope optics. For example, the aperture plane of the '705 patent pol-scope equipped with high angle (NA) lenses and crossed circular polarizers would, without polarization distortions, produce an aperture plane image that would be completely dark. Instead, towards the periphery the plane is brighter because light rays that pass through the periphery of the optical system pick up polarization distortions (Shribak, M., S. Inoué and R. Oldenbourg. 2002. Polarization aberrations caused by differential transmission and phase shift in high NA lenses: theory, measurement and rectification. Opt. Eng. 41: 943-954).